Citrus is susceptible to numerous disease caused by plant pathogens. Seven distinct viroid species representing four genera of the Pospiviroidae family have been identified: Citrus exocortis viroid (CEVd, genus Pospiviroid), Hop stunt viroid (HSVd, genus Hostuviroid), Citrus bark cracking viroid (CBCVd, genus Cocadviroid), and Citrus bent leaf viroid (CBLVd, genus As), Citrus dwarfing viroid (CDVd), Citrus viroid V (CVd-V) and Citrus viroid VI (CVd-VI).
Viroids are nonencapsidated, small circular, single-stranded RNAs that replicate autonomously when inoculated in their host plants. Most citrus viroids are widespread, usually occurring as complex mixtures that co-infect the same plant. Citrus viroids can elicit diseases in sensitive hosts such as exocortis and cachexia or affect tree size and crop (see, e.g., Semancik et al., J. Gen. Virol., 69:3059-3068 (1988); Semancik and Weathers, Virology, 46:456-466 (1972); and Verniere et al., Plant Dis., 88:1189-1197 (2004)).
Economic loss due to these diseases can be severe and is of particular concern in states such as California and Florida, which produce most of the United States' supply of citrus fruits. To prevent the spread of citrus disease, federal and state agencies have adopted registration programs that routinely performs time-consuming and costly pathogen tests to evaluate host resistance and viroid accumulation in plants and germplasm. Pathogen-infected germplasms must undergo treatment or removal from the certified registration programs regardless of the pathogen species, strain or isolate. Since agencies regularly screen thousands of samples, there remains an unmet need for an efficient and reliable method for the universal detection of all citrus viroid.
Currently, the most popular or standard molecular citrus viroid detection method is the conventional reverse transcription polymerase chain reaction (RT-PCR). Several RT-PCR protocols are available today for the detection of citrus viroids (Bernard, L. and Duran-Vila, N., Mol. Cell. Probes, 20:105-113 (2006); Ito et al., J. Virol. Methods, 106: 23.5-239 (2002)). However, in principal, the conventional RT-PCR requires seven different protocols with seven different sets of viroid specific primers for the detection of all known citrus viroids. In addition, methods such as gel electrophoresis are needed to visualize the results.
Nucleic acid-detection methods (e.g., microarray, real-time quantitative PCR (RT-qPCR)) have been used for real time detection of viral and bacterial pathogens (see, e.g., Aldea et al., J. Clin. Microbiol., 40:1060-2; Nadkarni et al., Microbiology, 148:257-66 (2002); Young et al., J. Virol. Methods, 103:27-39 (2002); Trottier et al., J. Virol. Methods, 103:89-99 (2002)). Real-time PCR technology allows for accurate quantitation of gene expression and gene expression patterns in multiple samples over a large dynamic range. The technology is used to analyze and indirectly quantitate mRNA expression levels by measuring the amount of amplified cDNA, rather than the amount of RNA in a sample. Real-time quantitative PCR is dependent on reverse transcription and PCR amplification, whereby a fluorescent signal incorporated into double-stranded DNA (dsDNA) is detected in each amplification cycle.
The present invention provides a method for the universal detection of all known citrus viroids using a multiplex of RT-qPCR assays. The present invention provides a universal, accurate, efficient, cost-effective, and quick method of detecting any citrus viroid pathogen. The method can be easily adapted for high throughput screenings.